Developments in meningococcal outer membrane vesicles

ABSTRACT

A first aspect of the invention provides meningococcal outer membrane vesicles in which NHBA is over-expressed. A second aspect of the invention provides meningococcal outer membrane vesicles in which NadA is over-expressed. A third aspect of the invention provides a panel of bacterial strains, each member of which is isogenic except for a single gene which in each strain encodes a different variant antigen of interest.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/822,238, which claims an international filing date of Sep. 9, 2011;which is the National Stage of International Application No.PCT/IB2011/053957. filed Sep. 9, 2011; which claims the benefit under 35U.S.C. §119(c) of U.S. Provisional Patent Application Nos. 61/381,859,filed Sep. 10,2010; and 61/429,673, filed Jan. 4, 2011, the disclosuresof which are herein incorporated by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 303822003801SeqList.txt,date recorded: Dec. 30, 2015, size: 102 KB).

FIELD OF THE INVENTION

This invention is in the field of meningococcal vaccines based onmembrane vesicles.

BACKGROUND

Various vaccines against serogroup B of Neisseria meningitidis (“MenB”)are currently being investigated. Some of these are based on outermembrane vesicles (OMVs), such as the Novartis MENZB™ product, theFinlay Institute VA-MENGOC-BC™ product, and the Norwegian Institute ofPublic Health MENBVAC™ product. Reference 1 discloses the constructionof vesicles from strains modified to express six different PorAsubtypes. References 2-4 report pre-clinical studies of an OMV vaccinein which fHbp (also known as GN1870) is over-expressed (and thisoverexpression can be combined with knockout of LpxL1 [5]). Reference 6recently reported a clinical study of five formulations of an OMVvaccine in which PorA & FrpB are knocked-out and Hsf & TbpA areover-expressed. Reference 7 reports a native outer membrane vesiclevaccine prepared from bacteria having inactivated synX, lpxL1, and lgtAgenes.

It is an object of the invention to provide further and improvedmeningococcal OMVs, and also to provide further and improvedmeningococci for use in vaccine production.

DISCLOSURE

A first aspect of the invention provides meningococcal outer membranevesicles in which NHBA is over-expressed. A second aspect of theinvention provides meningococcal outer membrane vesicles in which NadAis over-expressed. A third aspect of the invention provides a panel ofbacterial strains, each member of which is isogenic except for a singlegene which in each strain encodes a different variant of an antigen ofinterest.

Over-Expression

The first and second aspects of the invention provide meningococcalouter membrane vesicles in which certain antigens are over-expressed. Inthe first aspect, at least NHBA is over-expressed. In the second aspect,at least NadA is over-expressed. As discussed below, these vesicles areobtained from bacteria which over-express the relevant antigen(s). Thebacterium may express the antigen(s) already, but include a geneticmodification which, compared to a bacterium without that modification,increases expression of the antigen. This modification will usually beintroduced using recombinant techniques, such as site-directedmutagenesis or targeted homologous recombination, so vesicles of theinvention are usually obtained from recombinant bacteria. Typically abacterium will include (i) a gene under the control of a promoter withwhich it is not found in nature and/or (ii) a knockout of a gene whichis found in the bacterium in nature.

As a result of the over-expression, outer membrane vesicles preparedfrom the modified meningococcus contain higher levels of theover-expressed antigen(s). The increase in expression in the OMVs isusefully at least 10%, measured in mass of the relevant antigen per unitmass of OMV, and is more usefully at least 20%, 30%t 40%, 50%, 75%, 100%or more.

Suitable recombinant modifications which can be used to causeover-expression of an antigen include, but are not limited to: (i)promoter replacement; (ii) gene addition; (iii) gene replacement; or(iv) repressor knockout.

In promoter replacement, the promoter which controls expression of theantigen's gene in a bacterium is replaced with a promoter which provideshigher levels of expression. For instance, the gene might be placedunder the control of a promoter from a housekeeping metabolic gene. Inother embodiments, the antigen's gene is placed under the control of aconstitutive or inducible promoter. Similarly, the gene can be modifiedto ensure that its expression is not subject to phase variation. Methodsfor reducing or eliminating phase variability of gene expression inmeningococcus are disclosed in reference 8. These methods includepromoter replacement, or the removal or replacement of a DNA motif whichis responsible for a gene's phase variability.

In gene addition, a bacterium which already expresses the antigenreceives a second copy of the relevant gene. This second copy can beintegrated into the bacterial chromosome or can be on an episomalelement such as a plasmid. The second copy can have a stronger promotertitan the existing copy. The gene can be placed under the control of aconstitutive or inducible promoter. The effect of the gene addition isto increase the amount of expressed antigen. Where a plasmid is used, itis ideally a plasmid with a high copy number e.g. above 10, or evenabove 100.

In gene replacement, gene addition occurs but is accompanied by deletionof the existing copy of the gene. For instance, this approach was usedin reference 4, where a bacterium's endogenous chromosomal fHbp gene wasdeleted and replaced by a plasmid-encoded copy (see also reference 9).Expression from the replacement copy is higher than from the previouscopy, thus leading to over-expression.

In repressor knockout, a protein which represses expression of anantigen of interest is knocked out. Thus the repression does not occurand the antigen of interest can be expressed at a higher level.

Promoters for over-expressed genes can advantageously include a CREN[10].

A over-expressing modified strain will generally be isogenic with itsparent strain, except for a genetic modification. As a result of themodification, expression of the antigen of interest hi the modifiedstrain Is higher (under the same conditions) than in the parent strain.A typical modification will be to place a gene under the control of apromoter with which it is not found in nature and/or to knockout a genewhich encodes a repressor.

In embodiments where NHBA is over-expressed, various approaches can beused. For convenience, the approach already reported in reference 11 canbe used i.e. introduction of a NHBA gene under the control of anIPTG-inducible promoter. By this approach the level of expression ofNHBA can be proportional to the concentration of IPTG added to aculture. The promoter may include a CREN.

In embodiments where NadA is over-expressed, various approaches can beused. One useful approach involves deletion of the gene encoding NadR(NMB1843), which is a transcriptional repressor protein [12] whichdown-regulates or represses the NadA-encoding gene in all strainstested. Knockout of NadR results in high-level constitutive expressionof NadA. An alternative approach to achieve NadA over-expression is toadd 4-hydroxyphenylacetic to the culture medium. A further approach isto introduce a NadA gene under the control of an IPTG-induciblepromoter.

In some embodiments a bacterium over-expresses both NHBA and NadA.

In addition to over-expressing NHBA and/or NadA, a bacterium mayover-express one or more further antigens. For instance, a bacterium mayover-express one or more of: (a) NhhA; (b) TbpA; (c) HmbR; (d) Tbpb; (e)NspA; (f) Cu,Zn-superoxide dismutase; (g) Omp85; (h) App; and/or (i)fHbp. Over-expression of NhhA is already reported in references 6 and13. Over-expression of TbpA is already reported in references 13 and 14.Over-expression of HmbR is already reported in reference 15.Over-expression of TbpB is already reported in reference 14.Over-expression of NspA is already reported in reference 16, incombination with porA and cps knockout. Over-expression ofCu,Zn-superoxide dismutase is already reported in reference 14.Over-expression of fHbp is already reported in references 2-4 & 9, andby a different approach (expressing a constitutively-active mutant FNR)in references 17 & 18.

In some embodiments a bacterium over-expresses NHBA, NadA and fHbp.These three antigens are components of the “universal vaccine” disclosedin reference 19 or “4CMenB” [20,21]. In one embodiment, expression ofNHBA is controlled by a strong promoter, NadR is knocked out and thestrain expresses a constitutively active mutant FNR. In anotherembodiment, expression of NHBA is controlled by a strong promoter,expression of fHbp is controlled by a strong promoter, and NadR isknocked out. The bacterium can also be a bacterium which does notexpress an active MltA (GNA33), such that it spontaneously releasesvesicles which contain NHBA, NadA and fHbp. Ideally, the bacterium doesnot express a native LPS e.g. it has a mutant or knockout of LpxL1.

Vesicles

The first and second aspects of the invention provide meningococcalouter membrane vesicles. These outer membrane vesicles include anyproteoliposomic vesicle obtained by disruption of or blebbling from ameningococcal outer membrane to form vesicles therefrom that retainantigens from the outer membrane. Thus the term includes, for instance,OMVs (sometimes referred to as ‘blebs’), microvesicles (MVs [22]) and‘native OMVs’ (‘NOMVs’ [23]).

MVs and NOMVs are naturally-occurring membrane vesicles that formspontaneously during bacterial growth and are released into culturemedium. MVs can be obtained by culturing Neisseria in broth culturemedium, separating whole cells from the smaller MVs in the broth culturemedium (e.g. by filtration or by low-speed centrifugation to pellet wilythe cells and not the smaller vesicles), and then collecting the MVsfrom the cell-depleted medium (e.g. by filtration, by differentialprecipitation or aggregation of MVs, by high-speed centrifugation topellet the MVs). Strains for use in production of MVs can generally beselected on the basis of the amount of MVs produced in culture e.g.refs. 24 & 25 describe Neisseria with high MV production.

OMVs are prepared artificially from bacteria, and may be prepared usingdetergent treatment (e.g. with deoxycholate), or by non-detergent means(e.g. see reference 26). Techniques for forming OMVs include treatingbacteria with a bile acid salt detergent (e.g. salts of lithocholicacid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid,cholic acid, ursocholic acid, etc., with sodium deoxycholate [27 & 28]being preferred for treating Neisseria) at a pH sufficiently high not toprecipitate the detergent [29]. Other techniques may be performedsubstantially in the absence of detergent [26] using techniques such assonication, homogenisation, microfluidisation, cavitation, osmoticshock, grinding, French press, blending, etc. Methods using no or lowdetergent can retain useful antigens such as NspA [26]. Thus a methodmay use an OMV extraction buffer with about 0.5% deoxycholate or lowere.g. about 0.2%, about 0.1 %, <0.05% or zero.

A useful process for OMV preparation is described in reference 30 andinvolves ultrafiltration on crude OMVs, rather than instead of highspeed centrifugation. The process may involve a step ofultracentrifugation after the ultrafiltration takes place.

Another useful process for outer membrane vesicle production is toinactivate the mltA gene in a meningococcus, as disclosed in reference31. These mutant bacteria spontaneously release vesicles into theirculture medium.

If lipo-oligosaccharide (LOS) is present in a vesicle it is possible totreat the vesicle so as to link its LOS and protein components(“infra-bleb” conjugation [43]).

The vesicles may lack LOS altogether, or they may lack hexa-acylated LOSe.g. LOS in the vesicles may have a reduced number of secondary acylchains per LOS molecule [32]. For example, the vesicles may from astrain which has a lpxL1 deletion or mutation which results inproduction of a penta-acylated LOS [3,7]. LOS in a strain may lack alacto-N-neotetraose epitope e.g. it may be a lst and/or lgtB knockoutstrain [6], LOS may lack at least one wild-type primary O-linked fattyacid [33]. LOS having. The LOS may have no α chain. The LOS may compriseGlcNAc-Hep₂phosphoethanolamine-KDO₂-Lipid A [34].

The vesicles may include one, more than one, or (preferably) zero PorAserosubtypes. Modification of meningococcus to provide multi-PorA OMVsis known e.g. from references 1 and 35. Conversely, modification toremove PorA is also known e.g. from reference 6.

The vesicles may be free from one of both of PorA and FrpB. Preferredvesicles are PorA-free.

The invention may be used with mixtures of vesicles from differentstrains. For instance, reference 36 discloses vaccine comprisingmultivalent meningococcal vesicle compositions, comprising a firstvesicle derived from a meningococcal strain with a serosubtype prevalentin a country of use, and a second vesicle derived from a strain thatneed not have a serosubtype prevent in a country of use. Reference 37also discloses useful combinations of different vesicles. A combinationof vesicles from strains in each of the L2 and L3 immunotypes may beused in some embodiments.

Bacteria

As mentioned above, OMVs of the invention ate prepared from meningococciwhich over-express the relevant antigen(s) due to genetic modification.The invention also provides these bacteria. The bacteria can be used forpreparing OMVs of the invention.

In addition to genetic modification(s) which cause over-expression ofthe antigen(s) of interest, the bacteria may include one or more furthermodifications. For instance, the bacterium may have a knockout of one ormore of lpxL1, lgtB, porA, frpB, synX, lgtA, mltA and/or lst.

The bacterium may have low endotoxin levels, achieved by knockout ofenzymes involved in LPS biosynthesis [38,39].

The bacterium may be from any serogroup e.g. A, B, C, W135, Y. It ispreferably serogroup B.

The bacterium may be of any serotype (e.g. 1, 2a, 2b, 4, 14, 15, 16,etc.), any serosubtype, and any immunotype (e.g. L1; L2; L3; L3,3,7;L10; etc.). Vesicles can usefully be prepared from strains having one ofthe following subtypes: P1.2; P1.2.5; P1.4; P1.5; P1.5.2; P1.5.c;P1.5c,10; P1.7,16; P1.7,16b; P1.7h,4; P1.9; P1.15; P1.9,15; P1.12,13;P1.13; P1.14; P1.21,16; P1-22,14.

The bacterium may be from any suitable lineage, including hyperinvasiveand hypervirulent lineages e.g. any of the following seven hypervirulentlineages: subgroup I; subgroup III; subgroup IV-1; ET-5 complex; ET-37complex; A4 cluster; lineage 3. These lineages have been defined bymultilocus enzyme electrophoresis (MLEE), but multilocus sequence typing(MLST) has also been used to classify meningococci [ref. 40] e.g. theET-37 complex is the ST-11 complex by MLST, the ET-5 complex is ST-32(ET-5), lineage 3 is ST-41/44, etc.

In some embodiments a bacterium may include one or more of the knockoutand/or hyper-expression mutations disclosed in references 16 and 41-43.Suitable genes for modification include: (a) Cps, CtrA, CtrB, CtrC,CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PilC PorB,SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB [41]; (b) CtrA, CtrB, CtrC,CtrD, FrpB, GalE, HtrB/MsbB, LbpA, LbpB, LpxK, Opa, Opc, PhoP, PilC,PmrE, PmrE, SiaA, SiaB, SiaC, SiaD, TbpA, and/or TbpB; (c) ExbB, ExbD,rmpM, CtrA, CtrB, CtrD, GalE, LbpA, LpbB, Opa, Opc, PilC, PorB, SiaA,SiaB, SiaC, SiaD, TbpA, and/or TbpB; and (d) CtrA, CtrB, CtrD, FrpB,OpA, OpC, PilC, PorB, SiaD, SynA, SynB, and/or SynC.

A bacterium may have one or more, or all, of the followingcharacteristics: (i) down-regulated or knocked-out LgtB and/or GalE totruncate the meningococcal LOS: (ii) up-regulated TbpA; (iii)up-regulated NhhA; (iv) up-regulated Omp85; (v) up-regulated LbpA; (vi)up-regulated NspA; (vii) knocked-out PorA; (viii) down-regulated orknocked-out FrpB; (ix) down-regulated or knocked-out Opa; (x)down-regulated or knocked-out Opc; (xii) deleted cps gene complex, Atruncated LOS can be one that does not include asialyl-lacto-N-neotetraose epitope e.g. it might be agalactose-deficient LOS. The LOS may have no a chain.

Strain Production

The invention provides a process for preparing a meningococcal strainsuitable for OMV preparation, comprising steps of (i) choosing astarting strain which expresses a first amount of an antigen when grownin specific culture conditions, then (ii) modifying the starting strainto provide a modified strain, wherein the modified strain expresses asecond amount of the antigen when grown in the same specific cultureconditions, wherein the second amount is higher than the first amount;wherein the antigen is cither NHBA or NadA, The second amount of NHBA orNadA is usefully at least 10%, higher than the first amount, measured inmass of the relevant antigen per unit mass of bacteria, and is moreusefully at least 20%, 30%, 40%, 50%, 75%, 100% or more.

The invention provides a process for preparing a meningococcal strainsuitable for OMV preparation, comprising steps of (i) choosing astarting strain which expresses NHBA and/or NadA; and (ii) modifying thestarting strain to increase the amount of NHBA and/or NadA which itexpresses. The increased amount after modification in step (ii) isusefully at least 10%, higher than the first amount, measured in mass ofthe relevant antigen per unit mass of bacteria, and is more usefully atleast 20%, 30%, 40%, 50%, 75%, 100% or more.

Either of these processes can be followed by a step of (iii) culturingthe modified bacteria obtained in step (ii) to provide a bacterialculture.

In step (ii), the modification to increase expression of NHBA and/orNadA can be any of the modifications discussed above. For instance, thestrain can be modified by knocking out expression of NadR, therebyincreasing expression of NadA. The strain can also be modified toincrease or decrease expression of other polypeptides, as describedelsewhere herein e.g. to increase its fHbp expression, such as byintroducing a gene which encodes a constitutively-active mutant FNR.

The invention also provides a process for preparing a meningococcalvesicle, comprising a step of treating a bacterial culture obtained by aprocess of the invention (as described above) such that its outermembrane forms vesicles. This treatment step can use any of thetechniques discussed above.

The invention also provides a process for preparing a meningococcalvesicle, comprising a step of treating a meningococcus of the inventionsuch that its outer membrane forms vesicles. This treatment step can useany of the techniques discussed above.

Useful starting strains are in meningococcus serogroup B. Four usefulstarting meningococcal strains for preparing bacteria which over-expressan antigen of interest are MC58, NZ05/33, H44/76 and GB013. MC58 hasPorA serosubtype 1.7,16; NZ05/33 has serosubtype 1.7-2,4; H44/76 hasserosubtype 1.7,16; and GB013 has serosubtype 1.22,9.

Iogenic Panels

A third aspect of the invention provides a panel of bacterial strains(e.g. meningococci), each member of which is isogenic except for asingle gene which in each strain encodes a different variant of anantigen of interest. Thus the only genetic difference between eachmember of the panel is the coding sequence for this antigen. This panelcan be used to study the immunological effect of polymorphic forms of agene of interest found in different wild-type strains, without having toworry about variability due to differences in those strains which areunrelated to the antigen of interest. For instance, these panels can beused as test strains in a scrum bactericidal antibody assay to provide aconstant genetic background for assessing the cross-population killingof bacteria by antibodies which were raised against a specific sequencevariant.

A useful panel for an antigen of interest can be made be selecting astarting strain of meningococcus. A useful starting strain does notexpress the antigen of interest; if the starting strain does express theantigen of interest then expression of the endogenous gene can beknocked out e.g. by insertion of a marker gene. To create a panel, asite in the bacterial genome is chosen for insertion of a gene encodingthe antigen of interest. This site can be under the control of apromoter, such that different coding sequences can be introduced forexpression from this promoter, or it can lack a promoter, in which casethe introduced sequences should include a promoter. An important featureof the panel is that each member has the same promoter for expression ofthe antigen of interest, in the same location in the genome, such thatthe only genetic difference between them is the coding sequence for theantigen of interest.

The antigen of interest, which differs between panel members, can be anyuseful antigen which exists in polymorphic forms across a bacterialpopulation. Thus, for meningococcus, the antigen of interest could bee.g. fHbp, NadA, NHBA, Omp85, HmbR, NhhA, App, NspA, TbpA. etc.

The general approach of creating an isogenic panel for testing theeffect of sequence variability is not restricted to meningococcus andcan be used for any other bacterium.

Antigens

NHBA (Neisserial Heparin Binding Antigen)

NHBA [11] was included in the published genome sequence formeningococcal serogroup B strain MC58 [68] as gene NMB2132 (GenBankaccession number GI:7227388; SEQ ID NO: 9 herein). Sequences of NHBAfrom many strains have been published since then. For example, allelicforms of NHBA (referred to as protein '287) can be seen in FIGS. 5 and15 of reference 44, and in example 13 and FIG. 21 of reference 45 (SEQIDs 3179 to 3184 therein). Various immunogenic fragments of NHBA havealso been reported.

Preferred NHBA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 9; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 9, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14,16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 9.

The most useful NHBA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 9. Advantageous NHBAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

The NadA antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [68] as gene NMB1994 (GenBankaccession number GI:7227256; SEQ ID NO: 10 herein). The sequences ofNadA antigen from many strains have been published since then, and theprotein's activity as a Neisserial adhesion has been well documented.Various immunogenic fragments of NadA have also been reported.

Preferred NadA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 10; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 10, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 10.

The most useful NadA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 10. Advantageous NadAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject. SEQ IDNO: 6 is one such fragment.

HmbR

The full-length HmbR sequence was Included in the published genomesequence for meningococcal serogroup B strain MC58 [68] as gene NMB1668(SEQ ID NO: 7 herein). Reference 46 reports a HmbR sequence from adifferent strain (SEQ ID NO: 8 herein), and reference 15 reports afurther sequence (SEQ ID NO: 19 herein). SEQ ID NOs: 7 and 8 differ inlength by 1 amino acid and have 94.2% identity. SEQ ID NO: 19 is oneamino acid shorter titan SEQ ID NO; 7 and they have 99% identity (oneinsertion, seven differences) by CLUSTALW. The invention can use anysuch HmbR polypeptide.

The invention can use a polypeptide that comprises a full-length HmbRsequence, but it will often use a polypeptide that comprises a partialHmbR sequence. Thus in some embodiments a HmbR sequence used accordingto the invention may comprise an amino acid sequence having at least i %sequence identity to SEQ ID NO: 7, where the value of i is 50, 60, 70,80, 90, 95, 99 or more. In other embodiments a HmbR sequence usedaccording to the invention may comprise a fragment of at least jconsecutive amino acids from SEQ ID NO: 7, where the value of j is 7, 8,10, 12,14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more. In other embodiments a HmbR sequence used according tothe invention may comprise an amino acid sequence (i) having at least i% sequence identity to SEQ ID NO: 7 and/or (ii) comprising a fragment ofat least j consecutive amino acids from SEQ ID NO: 7.

Preferred fragments of j amino acids comprise an epitope from SEQ ID NO:7. Such epitopes will usually comprise amino acids that are located onthe surface of HmbR. Useful epitopes include those with amino acidsinvolved in HmbR's binding to haemoglobin, as antibodies that bind tothese epitopes can block the ability of a bacterium to bind to hosthaemoglobin. The topology of HmbR, and its critical functional residues,were investigated in reference 47. Fragments that retain a transmembranesequence are useful, because they can be displayed on the bacterialsurface e.g. in vesicles. Examples of long fragments of HmbR correspondto SEQ ID NOs: 21 and 22. If soluble HmbR is used, however, sequencesomitting the transmembrane sequence, but typically retaining epitope(s)from the extracellular portion, can be used.

The most useful HmbR antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 7. Advantageous HmbRantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

fHbp (Factor H Binding Protein)

The fHbp antigen has been characterised in detail. It has also beenknown as protein ‘741’ [SEQ IDs 2535 & 2536 in ref. 45], “NMB1870”,‘GNA1870’ [refs. 48-50], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [51-53]. It isnaturally a lipoprotein and is expressed across all meningococcalserogroups. The structure of fHbp's C-terminal immunodominant domain(‘fHbpC’) has been determined by NMR [54]. This part of the proteinforms an eight-stranded β-barrel, whose strands are connected by loopsof variable lengths. The barrel is preceded by a short α-helix and by aflexible N-terminal tail.

The fHbp antigen falls into three distinct variants [55] and it has beenfound that serum raised against a given family is bactericidal withinthe same family, but is not active against strains which express one ofthe other two families i.e. there is intra-family cross-protection, butnot inter-family cross-protection. The invention can use a single fHbpvariant, but is will usefully include a fHbp from two or three of thevariants. Thus it may use a combination of two or three different fHbps,selected from: (a) a first protein, comprising an amino acid sequencehaving at least a % sequence identity to SEQ ID NO: 1 and/or comprisingan amino acid sequence consisting of a fragment of at least x contiguousamino acids from SEQ ID NO: 1; (b) a second protein, comprising an aminoacid sequence having at least b % sequence identity to SEQ ID NO: 2and/or comprising an amino acid sequence consisting of a fragment of atleast y contiguous amino acids from SEQ ID NO: 2; and/or (c) a thirdprotein, comprising an amino acid sequence having at least c % sequenceidentity to SEQ ID NO: 3 and/or comprising an amino acid sequenceconsisting of a fragment of at least 2 contiguous amino acids from SEQID NO: 3.

The value of a is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, or more. The value of b is at least 85 e.g.86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 99.5, or more.The value of c is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98,99, 99.5, or more. The values of a, b and c are notintrinsically related to each other.

The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15,16, 17,18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y isat least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 225, 250). The values of x, y and z are not intrinsically relatedto each other.

Where the invention uses a single fHbp variant, a composition mayinclude a polypeptide comprising (a) an amino acid sequence having atleast a % sequence identity to SEQ ID NO: 1 and/or comprising an aminoacid sequence consisting of a fragment of at least x contiguous aminoacids from SEQ ID NO: 1; or (b) an amino acid sequence having at least b% sequence identity to SEQ ID NO: 2 and/or comprising an amino acidsequence consisting of a fragment of at least y contiguous amino acidsfrom SEQ ID NO: 2; or (c) an amino acid sequence having at least c %sequence identity to SEQ ID NO: 3 and/or comprising an amino acidsequence consisting of a fragment of at least z contiguous amino acidsfrom SEQ ID NO: 3.

Where the invention uses a fHbp from two or three of the variants, acomposition may include a combination of two or three different fHbpsselected from: (a) a first polypeptide, comprising an amino acidsequence having at least a % sequence identity to SEQ ID NO: 1 and/Ofcomprising an amino acid sequence consisting of a fragment of at least xcontiguous amino acids from SEQ ID NO: 1; (b) a second polypeptide,comprising an amino acid sequence having at least b % sequence identityto SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of afragment of at least y contiguous amino acids from SEQ ID NO: 2; and/or(c) a third polypeptide, comprising an amino acid sequence having atleast c % sequence identity to SEQ ID NO: 3 and/or comprising an aminoacid sequence consisting of a fragment of at least z contiguous aminoacids from SEQ ID NO: 3. The first, second and third polypeptides havedifferent amino acid sequences.

Where the invention uses a fHbp from two of the variants, a compositioncan include both: (a) a first polypeptide, comprising an amino acidsequence having at least a % sequence identity to SEQ ID NO: 1 and/orcomprising an amino acid sequence consisting of a fragment of at least xcontiguous amino acids from SEQ ID NO: 1; and (b) a second polypeptide,comprising an amino acid sequence having at least b % sequence identityto SEQ ID NO: 2 and/or comprising an amino acid sequence consisting of afragment of at least y contiguous amino acids from SEQ ID NO: 2. Thefirst and second polypeptides have different amino acid sequences.

Where the invention uses a fHbp from two of the variants, a compositioncan include both: (a) a first polypeptide, comprising an amino acidsequence having at least a % sequence identity to SEQ ID NO: 1 and/orcomprising an amino acid sequence consisting of a fragment of at least xcontiguous amino acids from SEQ ID NO: 1; (b) a second polypeptide,comprising an amino acid sequence having at least c % sequence identityto SEQ ID NO: 3 and/or comprising an amino acid sequence consisting of afragment of at least 2 contiguous amino acids from SEQ ID NO: 3. Thefirst and second polypeptides have different amino acid sequences.

Another useful fHbp which can be used according to the invention is oneof the modified forms disclosed, for example, in reference 56 comprisingSEQ ID NO: 20 or 23 therefrom. These modified forms can elicit antibodyresponses which are broadly bactericidal against meningococci.

fHbp protein(S) in a OMV will usually be lipidated at a N-terminuscysteine. In other embodiments they will not be lipidated.

The NspA antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [68] as gene NMB0663 (GenBankaccession number GI:7225888; SEQ ID NO: 11 herein). The antigen waspreviously known from references 57 & 58. The sequences of NspA antigenfrom many strains have been published since then. Various immunogenicfragments of NspA have also been reported.

Preferred NspA antigens for use with the invention comprise un aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 11; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 11, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 11.

The most useful NspA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 11. Advantageous NspAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

NhhA (Neisseria Hia Homologate)

The NhhA antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [68]as gene NMB0992 (GenBankaccession number GI:7226232; SEQ ID NO: 12 herein). The sequences ofNhhA antigen front many strains have been published since e.g. refs 44 &59, and various immunogenic fragments of NhhA have been reported. It isalso known as Hsf.

Preferred NhhA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 12; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 12, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 12.

The most useful NhhA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 12. Advantageous NhhAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

App (Adhesion and Penetration Protein)

The App antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [68] as gene NMB1985 (GenBankaccession number GI:7227246; SEQ ID NO: 13 herein). The sequences of Appantigen from many strains have been published since then. It has alsobeen known as ‘ORF1’ and ‘Hap’. Various immunogenic fragments of Apphave also been reported.

Preferred App antigens for use with the invention comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) toSEQ ID NO: 13; and/or (b) comprising a fragment of at least‘n’consecutive amino acids of SEQ ID NO: 13, wherein ‘n’ is 7 or more(e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90,100, 150, 200, 250 or more). Preferred fragments of (b) comprise anepitope from SEQ ID NO: 13.

The most useful App antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 13. Advantageous Appantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

Omp 85 (85 kDa Outer Membrane Protein)

The Omp85 antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [68] as gene NMB0182 (GenBankaccession number GI:7225401; SEQ ID NO: 14 herein). The sequences ofOmp85 antigen from many strains have been published since then. Furtherinformation on Omp85 can be found in references 60 and 61. Variousimmunogenic fragments of Omp85 have also been reported.

Preferred Omp85 antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 14; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 14, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 14.

The most useful Omp85 antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 14. Advantageous Omp85antigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

TbpA

The TbpA antigen was included in the published genome sequence formeningococcal serogroup 8 strain MC58 [68] as gene NMB0461 (GenBankaccession number GI:7225687; SEQ ID NO: 23 herein). The sequences ofTbpA from many strains have been published since then. Variousimmunogenic fragments of TbpA have also been reported.

Preferred TbpA antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 23; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 23, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 23.

The most useful TbpA antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 23. Advantageous TbpAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

TbpB

The TbpB antigen was included in the published genome sequence formeningococcal serogroup B strain MC58 [68] as gene NMB1398 (GenBankaccession number GI:7225686; SEQ ID NO: 24 herein). The sequences ofTbpB from many strains have been published since then. Variousimmunogenic fragments of TbpB have also been reported.

Preferred TbpB antigens for use with the invention comprise an aminoacid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 24; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 24, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 24.

The most useful TbpB antigens can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 24. Advantageous TbpBantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

Cu,Zn-Superoxide Dismutase

The Cu,Zn-superoxide dismutase antigen was included in the publishedgenome sequence for meningococcal serogroup B strain MC58 [68] as geneNMB1398 (GenBank accession number GI:7226637; SEQ ID NO: 25 herein). Thesequences of Cu,Zn-superoxide dismutase from many strains have beenpublished since then. Various immunogenic fragments of Cu,Zn-superoxidedismutase have also been reported.

Preferred Cu,Zn-superoxide dismutase antigens for use with the inventioncomprise an amino acid sequence: (a) having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5% or more) to SEQ ID NO: 25; and/or (b) comprising afragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 25,wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragmentsof (b) comprise an epitope from SEQ ID NO: 25.

The most useful Cu,Zn-superoxide dismutase antigens can elicitantibodies which, after administration to a subject, can bind to ameningococcal polypeptide consisting of amino acid sequence SEQ ID NO:25. Advantageous Cu,Zn-superoxide dismutase antigens for use with theinvention can elicit bactericidal anti-meningococcal antibodies afteradministration to a subject.

Pharmaceutical Compositions

Vesicles of the invention are useful as active ingredients inimmunogenic pharmaceutical compositions for administration to a patient.These will typically include a pharmaceutically acceptable carrier, anda thorough discussion of such carriers is available in reference 62.

Effective dosage volumes can be routinely established, but a typicalhuman dose of the composition has a volume of about 0.5 ml e.g. forintramuscular injection. The RIVM OMV-based vaccine was administered ina 0.5 ml volume [63] by intramuscular injection to the thigh or upperarm. MeNZB™ is administered in a 0.5 ml by intramuscular injection tothe anterolateral thigh or the deltoid region of the arm. Similar dosesmay be used for other delivery routes e.g. an intranasal OMV-basedvaccine for atomisation may have a volume of about 100 μl or about 130μl per spray, with four sprays administered to give a total dose ofabout 0.5 ml.

The pH of a composition of the invention is usually between 6 and 8, andmore preferably between 6.5 and 7.5 (e.g. about 7). The pH of the RIVMOMV-based vaccine is 7.4 [64], and a pH<7.5 is preferred forcompositions of the invention. The RIVM OMV-based vaccine maintains pHby using a 10 mM Tris/HCl buffer, and stable pH in compositions of theinvention may be maintained by the use of a buffer e.g. a Tris buffer, acitrate buffer, phosphate buffer, or a histidine buffer. Thuscompositions of the invention will generally include a buffer.

The composition may be sterile and/or pyrogen-free. Compositions of theinvention may be isotonic with respect to humans.

Compositions of the invention for administration to patients areimmunogenic, and are more preferably vaccine compositions. Vaccinesaccording to the invention may either be prophylactic (i.e. to preventinfection) or therapeutic (i.e. to treat infection), but will typicallybe prophylactic. Immunogenic compositions used as vaccines comprise animmunologically effective amount of antigen(s), as well as any othercomponents, as needed. By ‘immunologically effective amount’, it ismeant that the administration of that amount to an individual, either ina single dose or as part of a series, is effective for treatment orprevention. This amount varies depending upon the health and physicalcondition of the individual to be treated, age, the taxonomic group ofindividual to be treated (e.g. non-human primate, primate, etc.), thecapacity of the individual's immune system to synthesise antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.The antigen content of compositions of the invention will generally beexpressed in terms of the amount of protein per dose. A dose of about0.9 mg protein per ml is typical for OMV-based intranasal vaccines.

Compositions of the invention may include an immunological adjuvant.Thus, for example, they may include an aluminium salt adjuvant or anoil-in-water emulsion (e.g. a squalene-in-water emulsion). Suitablealuminium salts include hydroxides (e.g. oxyhydroxides), phosphates(e.g. hydroxyphosphates, orthophosphates), (e.g. see chapters 8 & 9 ofref. 65), or mixtures thereof. The stilts can take any suitable form(e.g. gel, crystalline, amorphous, etc.). with adsorption of antigen tothe salt being preferred. The concentration of Al⁺⁺⁺ in a compositionfor administration to a patient is preferably less than 5 mg/ml e.g. ≦4mg/ml, ≦3 mg/ml, ≦2 mg/ml, ≦1 mg/ml, etc. A preferred range is between0.3 and 1 mg/ml. A maximum of 0.85 mg/dose is preferred. Aluminiumhydroxide adjuvants are particularly suitable for use with meningococcalvaccines.

Meningococci affect various areas of the body and so the compositions ofthe invention may be prepared in various liquid forms. For example, thecompositions may be prepared as injectables, either as solutions orsuspensions. The composition may be prepared for pulmonaryadministration e.g. by an inhaler, using a fine spray. The compositionmay be prepared for nasal, aural or ocular administration e.g. as sprayor drops. Injectables for intramuscular administration are typical.Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format. Antimicrobials such as thiomersaland 2-phenoxyethanol are commonly found in vaccines, but it is preferredto use either a mercury-free preservative or no preservative at all.Compositions of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents ate generally present at lowlevels e.g. <0.01%.

Compositions of the invention may include residual detergent (e.g.deoxycholate) from OMV preparation. The amount of residual detergent ispreferably less than 0.4 μg (more preferably less than 0.2 μg) for everyμg of MenB protein.

If a composition of the invention includes LOS, the amount of LOS ispreferably less than 0.12 μg (more preferably less than 0.05 μg) forevery μg of protein.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical e.g. about 9 mg/ml.

Methods of Treatment

The invention also provides a method for raising an immune response in amammal, comprising administering a composition of the invention to themammal. The immune response is preferably protective and preferablyinvolves antibodies. The method may raise a booster response in apatient that has already been primed against N.meningitidis.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc.

The invention also provides vesicles of the invention for use as amedicament. The medicament is preferably used to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of vesicles of the invention in themanufacture of a medicament for raising an immune response in a mammal.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by A.meningitidis e.g. bacterial (or, morespecifically, meningococcal) meningitis, or septicemia.

One way of checking efficacy of therapeutic treatment involvesmonitoring Neisserial infection after administration of the compositionof the invention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against antigens afteradministration of the composition. Immunogenicity of compositions of theinvention can be determined by administering them to test subjects(e.g.. children 12-16 months age, or animal models [66]) thendetermining standard parameters including serum bactericidal antibodies(SBA) and ELISA litres (GMT). These immune responses will generally bedetermined around 4 weeks after administration of the composition, andcompared to values determined before administration of the composition.A SBA increase of at least 4-fold or 8-fold is preferred. Where morethan one dose of the composition is administered, more than onepost-administration determination may be made.

In general, compositions of the invention are able to induce serumbactericidal antibody responses after being administered to a subject.These responses are conveniently measured in mice and are a standardindicator of vaccine efficacy. Serum bactericidal activity (SBA)measures bacterial killing mediated by complement, and can be assayedusing human or baby rabbit complement WHO standards require a vaccine toinduce at least a 4-fold rise in SBA in more than 90% of recipients.MeNZB™ elicits a 4-fold rise in SBA 4-6 weeks after administration ofthe third dose.

Preferred compositions can confer an antibody titre in a human subjectpatient that is superior to the criterion for seroprotection for anacceptable percentage of subjects. Antigens with an associated antibodytitre above which a host is considered to be seroconverted against theantigen are well known, and such litres are published by organisationssuch as WHO. Preferably more than 80% of a statistically significantsample of subjects is seroconverted, more preferably more than 90%,still more preferably more than 93% and most preferably 96-100%.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by any other suitableroute. The invention may be used to elicit systemic and/or mucosalimmunity. Intramuscular administration to the thigh or the upper arm ispreferred, injection may be via a needle (e.g. a hypodermic needle), butneedle-free injection may alternatively be used. A typical intramusculardose is 0.5 ml.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined. The OMV-based RIVM vaccine was tested using a3- or 4-dose primary schedule, with vaccination at 0, 2 & 8 or 0, 1, 2 &8 months. MeNZB™ is administered sis three doses at six week intervals.

Compositions of the invention may be used to induce bactericidalantibody responses against more than one hypervirulent lineage ofmeningococcus. In particular, they can preferably induce bactericidalresponses against two or three of the following three hypervirulentlineages: (i) cluster A4; (ii) ET5 complex; and (iii) lineage 3. Theymay additionally induce bactericidal antibody responses against one ormore of hypervirulent lineages subgroup I, subgroup III, subgroup IV-Ior ET-37 complex, and against other lineages e.g. hyperinvasivelineages. This does not necessarily mean that the composition can inducebactericidal antibodies against each and every strain of meningococcuswithin these hypervirulent lineages e.g. rather, for any given group offour of more strains of meningococcus within a particular hypervirulentlineage, the antibodies induced by the composition ate bactericidalagainst at least 50% (e.g. 60%, 70%, 80%, 90% or more) of die group.Preferred groups of strains will include strains isolated in at leastfour of the following countries: GB, AU, CA, NO, IT, US, NZ, NL, BR, andCU. The serum preferably has a bactericidal titre of at least 1024 (e.g.2¹⁰, 2¹¹, 2¹², 2¹³, 2¹⁴, 2¹⁵, 2¹⁶, 2¹⁷, 2¹⁸ or higher, preferably atleast 2¹⁴) e.g. the serum is able to kill at least 50% of test bacteriaof a particular strain when diluted 1/1024.

Useful compositions can induce bactericidal responses against thefollowing strains of serogroup B meningococcus: (i) from cluster A4,strain 961-5945 (B:2b:P1.21,16) and/or strain G2136 (B:−); (ii) fromET-5 complex, strain MC58 (B:15:P1.7,16b) and/or strain 44/76(B:15:P1.7,16); (iii) from lineage 3, strain 394/98 (B:4:P1.4) and/orstrain BZ198 (B:NT:−). More preferred compositions can inducebactericidal responses against strains 961-5945, 44/76 and 394/98.

Strains 961-5945 and G2136 are both Neisseria MLST reference strains[ids 638 & 1002 in ref. 67]. Strain MC58 is widely available (e.g. ATCCBAA-335) and was the strain sequenced in reference 68. Strain 44/76 hasbeen widely used and characterised (e.g. ref. 69) and is one of theNeisseria MLST reference strains [id 237 in ref. 67; row 32 of Table 2in ref. 40]. Strain 394/98 was originally isolated in New Zealand in1998. and there have been several published studies using this strain(e.g. refs. 70 & 71). Strain BZ198 is another MLST reference strain (id409 in ref. 67; row 41 of Table 2 in ref. 40).

Further Antigenic Components

In addition to vesicles of the invention, an immunogenic composition caninclude further antigens.

In some embodiments, a composition includes one or more capsularsaccharides from meningococci e.g. from serogroups A, C, W135 and/or Y.These saccharides will usually be conjugated to a protein carrier. Acomposition of the invention may include one or more conjugates ofcapsular saccharides from 1, 2, 3, or 4 of meningococcal serogroups A,C, W135 and Y e.g. A+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135,A+C+Y, A+W135+Y, A+C+W135+Y, etc. Components including saccharides fromall four of serogroups A, C, W135 and Y are ideal.

As well as containing antigens from N.meningitidis, compositions mayinclude antigens from further pathogens. For example, the compositionmay comprise one or more of the following further antigens:

-   -   an antigen from Streptococcus pneumoniae, such as a saccharide        (typically conjugated)    -   an antigen from hepatitis B virus, such as the surface antigen        HBsAg.    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous hemagglutinin (FHA) from        B.pertussis, optionally also in combination with pertactin        and/or agglutinogens 2 and 3.    -   a diphtheria antigen, such as a diphtheria toxoid.    -   a tetanus antigen, such as a tetanus toxoid.    -   a saccharide antigen from Haemophilus influenzae B (Hib),        typically conjugated.    -   inactivated poliovirus antigens.

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens. DTP combinations are thus preferred.

If a Hib saccharide is included (typically as a conjugate), thesaccharide moiety may be a polysaccharide (e.g. full-lengthpolyribosylribitol phosphate (PRP) as purified from bacteria), but it isalso possible to fragment the purified saccharide to makeoligosaccharides (e.g. MW from ˜1 to ˜5 kDa) e.g. by hydrolysis. Theconcentration of Hib conjugate in a composition will usually be in therange of 0.5 μg to 50 μg e.g. from 1-20 μg, from 10-15 μg, from 12-16μg, etc. The amount may be about 15 g, or about 12.5 μg in someembodiments. A mass of less than 5 μg may be suitable [72] e.g. in therange 1-5 μg, 2-4 μg, or about 2.5 μg. As described above, incombinations that include Hib saccharide and meningococcal saccharides,the dose of the former may be selected based on the dose of the latter(in particular, with multiple meningococcal serogroups, their meanmass). Further characteristics of Hib conjugates are as disclosed abovefor meningococcal conjugates, including choice of carrier protein (e.g.CRM 197 or tetanus toxoid), linkages, ratios, etc.

If a S.pneumoniae antigen is included, this may be a polypeptide or asaccharide. Conjugates capsular saccharides are particularly useful forimmunising against pneumococcus. The saccharide may be a polysaccharidehaving the size that arises during purification of the saccharide frombacteria, or it may be an oligosaccharide achieved by fragmentation ofsuch a polysaccharide. In the 7-valent PREVNAR™ product, for instance, 6of the saccharides arc presented as intact polysaccharides while one(the 18C serotype) is presented as an oligosaccharide. A composition mayinclude a capsular saccharide from one or more of the followingpneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. A compositionmay include multiple serotypes e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes. 7-valent,9-valent, 10-valent, 11-valent and 13-valent conjugate combinations arealready known in the art, as is a 23-valent unconjugated combination.For example, an 10-valent combination may include saccharide fromserotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valentcombination may further include saccharide from serotype 3. A 12-valentcombination may add to the 10-valent mixture: serotypes 6A and 19A; 6Aand 22F; 19A and 22F; 6A and 15B; 19A and 15B; r 22F and 15B; A13-valent combination may add to the 11-valent mixture: serotypes 19Aand 22F; 8 and 12F; 8 and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12Fand 19A; 12F and 22F; 15B and 19A; 15B and 22F, etc. Furthercharacteristics of pneumococcal conjugates are as disclosed above formeningococcal conjugates, including choice of carrier protein (e.g. CRM197 or tetanus toxoid), linkages, ratios, etc. Where a compositionincludes more than one conjugate, each conjugate may use the samecarrier protein or a different carrier protein. Reference 73 describespotential advantages when using different carrier proteins inmultivalent pneumococcal conjugate vaccines.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references74-80, etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Where the invention concerns an “epitope”, this epitope may be a B-cellepitope and/or a T-cell epitope, but will usually be a B-ceil epitope.Such epitopes can be identified empirically (e.g. using PEPS CAN [81,82]or similar methods), or they can be predicted (e.g. using theJameson-Wolf antigenic index [83], matrix-based approaches [84],MAPITOPE [85], TEPITOPE [86,87], neural networks [88], OptiMer & EpiMer[89, 90], ADEPT [91], Tsites [92], hydrophilicity [93], antigenic index[94] or the methods disclosed in references 95-99. etc.). Epitopes arethe parts of an antigen that are recognised by and bind to the antigenbinding sites of antibodies or T-cell receptors, and they may also bereferred to as “antigenic determinants”.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.100. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2. BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. 101.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the approach for constructing an isogenic panel byknocking out nadA and nhba (GNA2132) to create a background strain.

FIG. 2 shows the insertion of fHbp genes into the background strain tomake a panel of isogenic strains expressing different fHbp genes underthe control of a Ptac promoter.

FIG. 3A-FIG. 3B shows expression levels of fHbp in the isogenic panelstrains described in FIG. 2.

FIG. 4A-FIG. 4B shows expression of NadA (upper panel) and NadR (lowerpanel) in eight wild-type strains (FIG. 4A) or their NadR knockout forms(FIG. 4B). The numbers in FIG. 4A show the number of TAAAtetranucleotide repeats in the strain. FIG. 4C-FIG. 4D shows expressionof NadA and NadR in 7 strains, in the presence of absence of 4HPA.

FIG. 5A-FIG. 5C shows (FIG. 5A) starting strain MC58 (FIG. 5B) MC58Δnhbaand (FIG. 5C) MC58Δnhba transformed with a complementing nhba gene withan upstream CREN and IPTG-inducible promoter.

FIG. 6 shows NHBA expression by MC58 and derivative strains. The lefttwo lanes show expression in MC58 and MC58Δnhba. The next 8 lanes showexpression in complemented strains at four concentrations of IPTG. Thelanes are arranged in pairs, with the right-hand lane being a straincomplemented with nhba having an upstream CREN.

FIG. 7 shows NHBA expression by 95N477 and derivative strains. The lefttwo lanes show expression in 95N477 and 95N477Δnhba. The next 5 lanesshow expression in complemented strains at the indicated concentrationsof IPTG.

FIG. 8 shows NHBA expression for five strains in an isogenic panel. Fromtop to bottom the expressed NHBA is from strain NZ98/254, UK013, UK355,2996 and NM117.

MODES FOR CARRYING OUT THE INVENTION NHBA

The endogenous nhba gene is knocked out in various serogroup B strainsto create strains MC58ΔΔnhba, 95N477Δnhba, NGH38Δnhba and UK013Δnhba.These strains arc then transformed with pCOMPpind-287 vector containinga gene encoding nhba from strain 394/98, with or without an upstreamCREN (contact regulatory element of Neisseria), under the control of anIPTG-inducible promoter. The vectors insert the nhba gene (±CREN)between the endogenous nmb1428 and nmb1429 genes by homologousrecombination.

FIG. 5 shows the starting MC58 strain, the MC58Δnhba strain, and thecomplemented MC58 strain (+CREN). FIG. 6 shows expression of NHBA by thevarious MC58 strains with increasing IPTG concentration. Thecomplemented strains show high levels of inducible NHBA expression, withthe highest levels seen with the inserted gene has an upstream CREN.

FIG. 7 shows expression in the 95N477 strains. The endogenous nhba genein this strain encodes a 427aa protein, whereas the insertedcomplementing gene has 492aa. Increased expression levels of the largerNHBA protein are clearly visible, and this expression increases withIPTG concentration.

Although in some strains (e.g. M4407) it was not possible to obtain aΔnhba knockout using the transformation protocols, for strains whichcould be transformed these results show that strains which over-expressNHBA can readily be obtained.

NadR (NMB 1843)

The nadA gene is present in approximately 50% of meningococcal isolates.NadA exhibits growth-phase dependent expression, with maximal levels inthe stationary growth phase of all strains tested. Expression iscontrolled by a tetranucleotide repeat (TAAA) located upstream of thenadA promoter. The number of repeats can be modified during replicationthrough slipped strand mispairing, and consequently can influence theexpression of the nadA gene by creating variants where changes in therepeat number result in promoters with low, medium or high activity.

An area of the nadA promoterm upstream of the TAAA repeat, isresponsible for repression of nadA expression during logarithmic phaseof growth. This area is called the ‘PR region’. DNA-affinityfractionation identified a protein present in meningococcus crudeextracts which binds to the GPR region. This protein is NadR (NMB1843)and is a member of the MarR family of repressors. NadR binds to threeoperators (binding sites) in the nadA promoter and results in repressionof NadA expression. Knockout of NadR in strains expressing high, mediumor low levels of NadA results in almost comparable high level expressionin each strain. Thus NadR is the repressor that contributes to thedifferential expression levels exhibited by meningococcal strains, orphase variants in the same strain, with different numbers of repeats intheir promoter. NadR is expressed to similar levels in different strainsbut can repress more or less efficiently the nadA promoter depending onthe number of repeats present in the variant promoter.

Knockout of NadR in various meningococcus backgrounds results in almostcomparable high levels of expression of NadA across the panel. Strainsate transformed with the knockout construct for the allelic replacementof nmb1843 with a chloramphenicol cassette. Expression levels in eightdifferent strains arc shown in FIG. 4.

A small molecule ligand 4-hydroxyphenylacetic acid (4HPA) can induceNadA expression in vitro due to derepression of NadR (FIG. 4C). Additionof the molecule to the purified NadR protein in vitro can inhibit thebinding activity of the protein for the nadA promoter. 4HPA is ametabolite of the catabolic pathway of the aromatic amino acids and issecreted in human saliva and urine, and so in vivo expression of NadAmay be higher than is seen during in vitro growth.

Thus strains which over-express NadA can readily be obtained byinactivation of NadR and/or by addition of a small molecule inducer tothe growth medium.

Isogenic Panel—NHBA

NHBA is an antigen in the 4CMenB product. An isogenic panel was used tostudy the potential cross protection of NHBA-induced bactericidalantibodies.

The nhba genes from six different meningococcal strains were amplifiedto provide the mature form of the polypeptide with a C-terminishistidine tag. These were cloned into the pET-21b+ plasmid vector andexpressed in E.coli. The purified NHBA peptides were then used toimmunize mice (20 μg dose) and obtain mouse antisera. ELISA assays wereperformed in order to confirm the presence of antibodies in ail themouse sera obtained.

To evaluate the immunogenicity and the contribution of amino acidsequence variability to vaccine coverage, a starting strain wasengineered to be susceptible to bactericidal killing only by anti-NHBAantibodies (rather than the other antigens in 4CMenB). N.meningitidisstrain 5/99 naturally expresses high levels of NadA, but very low levelsof NHBA and fHbp. Its nadA and nhba genes were respectively replaced byery and kan resistance cassettes (5/99AA). The nhba gene to becomplemented was then inserted in the intergenic region between the openreading frames nmb1428 and nmb1429. Thus the final strain panel wasisogenic except for the chosen nhba gene, and this gene should beinducable for expression at equal levels in all members of the panel.

FACS showed that the panel members showed a comparable amount of thedifferent NHBA polypeptides in each strain (FIG. 8). Several mouseantisera raised against the different NHBA polypeptides were tested inwestern blot and the detection appeared to be variant-specific, showinga stronger recognition for the homologous variant.

The panel was also used for testing the bactericidal effect of the mouseantisera. As the strains were isogenic then any difference inbactericidal effect should arise only from the different expressed NHBApolypeptides. In parallel the sera were tested against wild-type strainswhich express the relevant NHBA polypeptide sequence, to see if thecommon genetic background of the isogenic panel did enable the detectionof differences which would be concealed by natural variation ifwild-type strains were used. Results were as follows:

Antiserum NHBA 5/99ΔΔ Wild-type NZ98/254 >8192 8192 MC58 8192 512 UK013256 128 UK355 128 256 2996 128 256 NM117 2048 4096

Thus the panel does seem to compensate for variability which isunrelated to the NHBA antigen itself. For instance, serum raised againstthe MC58 sequence is much more effective against the MC58 polypeptide inthe isogenic panel than against the wild-type MC5B strain.

Isogenic Panel—fHbp

Sequencing of the fHbp gene in a large collection of meningococcalisolates revealed three variants with low levels of cross-protectivebactericidal response. A serum bactericidal assay was used to evaluatethe cross-protective capabilities of human antibodies raised againstdifferent fHbp variants, but the killing mediated by bactericidalantibodies in this assay is dependent by several factors. Thus thepotential coverage of a single antigen may be difficult to estimate.

A genetic approach was used to overcome variability due tostrain-specific serum susceptibility, limitations of compatiblecomplement sources, and variable expression of fHbp and othersurface-exposed factors affecting resistance to serum (e.g. thecapsule). A well-characterized meningococcal isolate (5/99) wasengineered to generate isogenic strains expressing ten different fHbpsub-variants from a constitutive heterologous promoter. The fHbp geneswere inserted between endogenous nmb1428 and nmb1429 genes. This panelwas then used as the test strain in a serum bactericidal antibody (SBA)assay to assess the ability of a single fHbp variant to elicit abroadly-protective immune response.

In order to have a genetic background to express different fHbpsub-variants without the interfering action of the other antigens, thenadA and nhba genes in the starting 5/99 strain were inactivated byinsertion of erm and kan resistance cassettes, respectively (FIG. 1).The resulting double mutant strain (named 5/99ΔΔ) was manipulated toexpress different fHbp subvariants under the control of a Ptac promoterto standardize the amount of fHbp expressed (FIGS. 1 & 2). In total, Jendifferent fHbp coding sequences were cloned in the pComp-RBS vector andtransferred to the 5/99ΔΔ genetic background.

To evaluate the expression of fHbp in the recombinant strains, weperformed FACS analysis using a mouse polyclonal scrum against a singlefHbp variant. The analysis showed a comparable amount of the differentfHbp sub-variants on the surface of the recombinant strains generated(FIG. 3). The recombinant strains were analyzed for their susceptibilityto killing by bactericidal antibodies from mice in a SBA using rabbitcomplement. Pooled sera from mice immunized with the “universal vaccine”of reference 19 or with its GNA2091-fHbp component were tested for theirability to kill the 5/99 wild-type, the intermediate 5/99ΔΔ strainexpressing neither NHBA nor NadA antigens, and the ten recombinantstrains. The 5/99 strain was killed by sera raised against the universalvaccine, but not by sera raised against the single antigen GNA2091-fHbp.The 5/99ΔΔ strain was resistant to killing by all sera. All of thecomplemented strains except one showed significant susceptibility tosera derived from mice immunized with the universal vaccine or withGNA2091-fHbp antigen. The single surviving strain expressed a fHbp infamily III, confirming the absence of cross-reactivity between the fHbpfamilies. The nine susceptible strains confirm that the specific fHbpsequence in the universal vaccine can raise antibodies which are broadlyprotective across the whole of fHbp family I.

The panel was also tested using sera obtained from human adults who wereimmunised with 4CMenB. The results were comparable to those seen usingmice.

It will be understood that the invention is described above by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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We claim:
 1. A modified meningococcus bacterium which over-expressesNadA and/or NHBA.
 2. The modified meningococcus bacterium of claim 1,which also over-expresses fHbp.
 3. The modified meningococcus bacteriumof claim 1, wherein the bacterium is isogenic with the unmodifiedmeningococcus bacterium, except for a genetic modification which causesthe bacterium to express more NadA and/or NHBA than the unmodifiedmeningococcus bacterium.
 4. The modified meningococcus bacterium ofclaim 3, which includes (i) a gene under the control of a promoter whichdoes not control that gene in the unmodified meningococcus bacteriumand/or (ii) a knockout of a gene which is found in the unmodifiedmeningococcus bacterium.
 5. The modified meningococcus bacterium ofclaim 1, wherein expression of NHBA is controlled by an inducible orconstitutive promoter, and wherein the promoter optionally includes aCREN.
 6. The modified meningococcus bacterium of claim 1, wherein thebacterium does not express NadR.
 7. The modified meningococcus bacteriumof claim 1, wherein the bacterium also expresses more fHbp than theparental strain.
 8. The modified meningococcus bacterium of claim 1,wherein expression of NHBA is controlled by a strong promoter, NadR isknocked out, and the strain expresses a constitutively active mutantFNR.
 9. The modified meningococcus bacterium of claim 1, whereinexpression of NHBA is controlled by a strong promoter, expression offHbp is controlled by a strong promoter, and NadR is knocked out. 10.The modified meningococcus bacterium of claim 1, wherein the bacteriumhas a knockout of LpxL1.
 11. The modified meningococcus bacterium ofclaim 1, wherein the bacterium does not express an active MltA.
 12. Themodified meningococcus bacterium of claim 1, wherein the bacterium doesnot express PorA.
 13. The modified meningococcus bacterium of claim 1,wherein the bacterium does not express FrpB.
 14. The modifiedmeningococcus bacterium of claim 1, wherein the bacterium is serogroupB.
 15. The modified meningococcus bacterium of claim 1, where thebacterium is immunotype L3.
 16. A process for preparing the modifiedmeningococcus bacterium of claim 1, suitable for OMV preparation,comprising steps of (i) choosing a starting strain which expresses NHBAand/or NadA; and (ii) modifying the starting strain to increase theamount of NHBA and/or NadA which it expresses.
 17. A process forpreparing a modified meningococcus bacterium suitable for OMVpreparation, comprising steps of (i) choosing a starting meningococcusbacterium which expresses a first amount of NHBA when grown in specificculture conditions, then (ii) modifying the starting meningococcusbacterium to provide the modified meningococcus bacterium, wherein themodified meningococcus bacterium expresses a second amount of NHBA whengrown in the same specific culture conditions, wherein the second amountis higher than the first amount.
 18. The process of claim 17, includinga step (iii) culturing the modified meningococcus bacterium obtained instep (ii) to provide a bacterial culture.
 19. A process for growingmeningococcus bacteria, wherein the bacteria are grown in the presenceof 4-hydroxyphenylacetic acid (or an analogue thereof which can induceNadA expression).
 20. A process for preparing a meningococcal vesicle,comprising a step of treating a bacterial culture comprising themodified meningococcus bacterium produced by the process of claim 18such that its outer membrane forms vesicles.
 21. A method for raising animmune response in a mammal, comprising administering a compositioncomprising meningococcal vesicles produced by the process of claim 20 tothe mammal.